Oligonucleotides and their analogs have been developed and used in molecular biology in a variety of procedures as probes, primers, linkers, adapters, and gene fragments. Modifications to oligonucleotides used in these procedures include labeling with nonisotopic labels, e.g. fluorescein, biotin, digoxigenin, alkaline phosphatase, or other reporter molecules. Other modifications have been made to the ribose phosphate backbone to increase the nuclease stability of the resulting analog. Examples of such modifications include incorporation of methyl phosphonate, phosphorothioate, or phosphorodithioate linkages, and 2′-O-methyl ribose sugar units. Further modifications include those made to modulate uptake and cellular distribution. With the success of these compounds for both diagnostic and therapeutic uses, there exists an ongoing demand for improved oligonucleotides and their analogs.
It is well known that most of the bodily states in multicellular organisms, including most disease states, are effected by proteins. Such proteins, either acting directly or through their enzymatic or other functions, contribute in major proportion to many diseases and regulatory functions in animals and man. For disease states, classical therapeutics has generally focused upon interactions with such proteins in efforts to moderate their disease-causing or disease-potentiating functions. In newer therapeutic approaches, modulation of the actual production of such proteins is desired. By interfering with the production of proteins, the maximum therapeutic effect may be obtained with minimal side effects. It is therefore a general object of such therapeutic approaches to interfere with or otherwise modulate gene expression, which would lead to undesired protein formation.
One method for inhibiting specific gene expression is with the use of oligonucleotides, especially oligonucleotides which are complementary to a specific target messenger RNA (mRNA) sequence. Several oligonucleotides are currently undergoing clinical trials for such use. Phosphorothioate oligonucleotides are presently being used as such antisense agents in human clinical trials for various disease states, including use as antiviral agents.
Transcription factors interact with double-stranded DNA during regulation of transcription. Oligonucleotides can serve as competitive inhibitors of transcription factors to modulate their action. Several recent reports describe such interactions (see Bielinska, A., et. al., Science, 1990, 250, 997-1000; and Wu, H., et. al., Gene, 1990, 89, 203-209).
In addition to such use as both indirect and direct regulators of proteins, oligonucleotides and their analogs also have found use in diagnostic tests. Such diagnostic tests can be performed using biological fluids, tissues, intact cells or isolated cellular components. As with gene expression inhibition, diagnostic applications utilize the ability of oligonucleotides and their analogs to hybridize with a complementary strand of nucleic acid. Hybridization is the sequence specific hydrogen bonding of oligomeric compounds via Watson-Crick and/or Hoogsteen base pairs to RNA or DNA. The bases of such base pairs are said to be complementary to one another.
Oligonucleotides and their analogs are also widely used as research reagents. They are useful for understanding the function of many other biological molecules as well as in the preparation of other biological molecules. For example, the use of oligonucleotides and their analogs as primers in PCR reactions has given rise to an expanding commercial industry. PCR has become a mainstay of commercial and research laboratories, and applications of PCR have multiplied. For example, PCR technology now finds use in the fields of forensics, paleontology, evolutionary studies and genetic counseling. Commercialization has led to the development of kits which assist non-molecular biology-trained personnel in applying PCR oligonucleotides and their analogs, both natural and synthetic, are employed as primers in such PCR technology.
Oligonucleotides and their analogs are also used in other laboratory procedures. Several of these uses are described in common laboratory manuals such as Molecular Cloning, A Laboratory Manual, Second Ed., J. Sambrook, et al., Eds., Cold Spring Harbor Laboratory Press, 1989; and Current Protocols In Molecular Biology, F. M. Ausubel, et al., Eds., Current Publications, 1993. Such uses include as synthetic oligonucleotide probes, in screening expression libraries with antibodies and oligomeric compounds, DNA sequencing, in vitro amplification of DNA by the polymerase chain reaction, and in site-directed mutagenesis of cloned DNA. See Book 2 of Molecular Cloning, A Laboratory Manual, supra. See also “DNA-protein interactions and The Polymerase Chain Reaction” in Vol. 2 of Current Protocols In Molecular Biology, supra.
Oligonucleotides and their analogs can be synthesized to have customized properties that can be tailored for desired uses. Thus a number of chemical modifications have been introduced into oligomers to increase their usefulness in diagnostics, as research reagents and as therapeutic entities. Such modifications include those designed to increase binding to a target strand (i.e. increase their melting temperatures, Tm), to assist in identification of the oligonucleotide or an oligonucleotide-target complex, to increase cell penetration, to stabilize against nucleases and other enzymes that degrade or interfere with the structure or activity of the oligonucleotides and their analogs, to provide a mode of disruption (terminating event) once sequence-specifically bound to a target, and to improve the pharmacokinetic properties of the oligonucleotide.
The chemical literature discloses numerous well-known protocols for coupling nucleosides through phosphorous-containing covalent linkages to produce oligonucleotides of defined sequence. One of the most routinely used protocols is the phosphoramidite protocol (see, e.g., Advances in the Synthesis of Oligonucleotides by the Phosphoramidite Approach, Beaucage, S. L.; Iyer, R. P., Tetrahedron, 1992, 48, 2223-2311 and references cited therein), wherein a nucleoside or oligonucleotide having a free hydroxyl group is reacted with a protected cyanoethyl phosphoramidite monomer in the presence of a weak acid to form a phosphite-linked structure. Oxidation of the phosphite linkage followed by hydrolysis of the cyanoethyl group yields the desired phosphodiester or phosphorothioate linkage.
Phosphoramidites are commercially available from a variety of commercial sources (included are: Glen Research, Sterling, Va.; Amersham Pharmacia Biotech Inc., Piscataway, N.J.; Cruachem Inc., Aston, Pa.; Chemgenes Corporation, Waltham, Mass.; Proligo LLC, Boulder, Colo.; PE Biosystems, Foster City Calif.; Beckman Coulter Inc., Fullerton, Calif.). Purity of commercially available phosphoramidites are generally around 98 or 99%. Some commercial sources set a standard and claim that all phosphoramidites are equal to or higher purity than the set standard. Alternatively, some commercial sources include assay results for each batch of phosphoramidites sent out. Commercially available phosphoramidites are prepared for the most part for automated DNA synthesis and as such are prepared for immediate use for synthesizing desired sequences of oligonucleotides. The high purity has been taught in the art to be a requisite to adequate coupling efficiency during synthesis.
A representative example of the rigorous quality assurance criteria applied to commercially available phosphoramidites is illustrated in the Glen Research Catalog. After synthesis and purification each phosphoramidite is subjected to quality assurance tests. Included in these tests is HPLC purification which results are used to establish the identity and purity of the particular amidite. Further tests include TLC, 31P NMR, a coupling test, a solution test, and a loss on drying test. The threshold purity of these amidites from this supplier is set at greater than 98%. The manufacturer further determines by 31P NMR that aside from the phosphoramidite no other phosphorus species are present.
Oligonucleotide synthesis has evolved as a practice that has been reported to required an almost antiseptic environment to obtain the desired product or a reasonable amount of product having a reasonable purity. This dogma has been stressed in a number of books dedicated to the synthesis of oligonucleotides and analogs. In Oligonucleotides and Analogues, A Practical Approach, Eckstein Ed, IRL Press; New York, 1991; the coupling efficiency e.g. average stepwise yield, is stated to be the primary factor in the over yield and purity of a product oligonucleotide. For example it is shown that a coupling efficiency of 99.5% will give a 90.9% overall yield for the synthesis of a 20-mer. If the coupling efficiency is reduced to only 90% the overall yield of the 20-mer drops to 13.5%. Hence, it can be seen that the coupling efficiency is very controlling, especially for oligonucleotides greater than about a 10-mer (38.7% overall yield with a 90% coupling efficiency). It is further stated that a coupling efficiency of less than 98% is totally unacceptable for routine oligonucleotide synthesis of longer sequences. Another factor that results directly from a low yield is that purification of the final oligonucleotide becomes much more difficult and costly. To achieve a good average coupling efficiency the use of good quality reagents is stressed.
Oligonucleotide Synthesis, a Practical Approach, Gait Ed, IRL Press; New York, 1984; is another publication detailing the synthesis of oligonucleotides. It also stresses the importance of using “the highest purity of batches of reagents”. The effect of impurities at each coupling is stated to be accumulative requiring removal from the final oligonucleotide after synthesis. It is further stated that the purification of such impure oligonucleotides often require two separate purification procedures to give the desired purity.
The synthesis of oligonucleotides has classically involved obtaining a desired product which was in itself a challenge. The synthesis of oligonucleotides has more recently evolved to the point that routine syntheses are being performed on kilogram scale. Moving forward the next step is the synthesis of oligonucleotides and analogs on ton scale. The evolution of oligonucleotide synthetic techniques toward large scale is requiring a reevaluation of each aspect of the synthetic process. One such aspect is the cost of the phosphoramidites used in oligonucleotide synthesis.
Commercially available high purity phosphoramidites generally account for about 40% of the overall cost of oligonucleotide synthesis. This 40% reflects the synthesis of the phosphoramidite and the subsequent purification and analysis of the phosphoramidite prior to sale. A reduction in the cost of phosphoramidites could have a significant effect on the cost of oligonucleotides produced therefrom. Consequently, there remains a need in the art for synthetic methods that will overcome these problems.
Several processes are known for the solid phase synthesis of oligonucleotide compounds. These are generally disclosed in the following U.S. Pat. No. 4,458,066; issued Jul. 3, 1984; U.S. Pat. No. 4,500,707, issued Feb. 19, 1985; and U.S. Pat. No. 5,132,418, issued Jul. 21, 1992. Additionally, a process for the preparation of oligonucleotides using phosphoramidite intermediates is disclosed in U.S. Pat. No. 4,973,679, issued Nov. 27, 1990.
A process for the preparation of phosphoramidites is disclosed in U.S. Pat. No. 4,415,732, issued Nov. 15, 1983.
Phosphoramidite nucleoside compounds are disclosed in U.S. Pat. No. 4,668,777, issued May 26, 1987.
A process for the preparation of oligonucleotides using a β-eliminating phosphorus protecting group is disclosed in U.S. Pat. No. Re. 34,069, issued Sep. 15, 1992.
A process for the preparation of oligonucleotides using a β-eliminating or allylic phosphorus protecting group is disclosed in U.S. Pat. No. 5,026,838, issued Jun. 25, 1991.